This invention relates to inhibition of HIV-1 replication using antisense RNA expression.
1. Field of the Invention
2. Description of the Related Art
HIV-1 infection is believed to be the primary cause of Acquired Immunodeficiency Syndrome (AIDS). HIV-1 is a retrovirus having a genome comprised of two copies of full length RNA. Without intending to be bound by a particular theory, it is hypothesized that the replication of the virus in the CD4+ host cell occurs as follows. When the host cell is infected, the viral genomic RNA is transcribed by reverse trascriptase into double stranded DNA. This double stranded DNA is then integrated into the host cell""s chromosome(s). When this double stranded DNA is integrated iinto the genetic material of the host cell, it is called a provirus. Following activation of the host cell, the provirus is transcribed into RNA in two distinct phases. In the early phase of infection, RNA transcripts of the provirus produced in the nucleus are converted into multiple copies of short sequences by cellular splicing enzymes. These short RNA transcripts encode genes for proteins, e.g., tat, which regulate the further transcription, and rev, which is though to mediate the transition into the late phase transcription. This early phase dominates for about 24 hours. About 24 hours after activation of the cell, the transcription moves into the late phase. In late phase transcription, long unspliced RNA transcripts of about 9,200 bases and medium-length single-spliced transcripts of about 4,500 bases move out of the nucleus and into the cytoplasm. These unspliced and single-spliced transcripts encode the structural and enyzmatic proteins of the virus. These unspliced and single-spliced transcripts include, inter alia, the following regions: gag, which encodes the viral core proteins; pol, which encodes various enzymes; and env, which encodes the two envelope proteins. FIG. 4 depicts the HIV-1 genomic structure. It will be noted that there is some overlap in the genes, because certain genes share some base sequences.
The unspliced and single-spliced transcripts are then further spliced, and the resulting mRNA is translated to produce the proteins necessary to make a new virus. The gag and pol regions are translated to produce the polyproteins gag and gag-pol, which are then cleaved by protease to form the mature proteins found in the virus. The env is spliced to generate a subgenomic messenger which encodes for the env polyproteins, which is likewise cleaved to produce the mature envelope proteins. Two strands of the viral RNA are then packaged into a core and surrounded with capsid protein, and the resulting virus is released from the cell together with a portion of the cell membrane.
Various antisense strategies to inhibit HIV-1 infection have been tried, including the use of trans-domninant proteins (Bevec, D., et al. 1992. Inhibition of human immunodeficiency virus type 1 replication in human T cells by retroviral-mediated gene transfer of a dominant-negative rev trans-activator. Proc. Natl. Acad. Sci. USA 89:9870-9874 and Trono, D., et al. 1989. HIV-1 gag mutants can dominantly interfere with the replication of the wild-type virus. Cell 59:113-120), single chain antibodies (Levy-Mintz, P., et al. 1996. Intracellular expression of single-chain variable fragments to inhibit early stages of the viral life cycle by targeting human immunodeficiency virus type 1 integrase. J. Virol. 70:8821-8832.), antisense RNAs (Chatterjee, S., et al. 1992. Dual-target inhibition of HIV-1 in vitro by means of adeno-associated virus antisense vector. Science 258:1485-1488., Choli, H., et al. 1994. Inhibition of HIV-1 multiplication in a human CD4+ lymphocytic cell line expressing antisense and sense RNA molecules containing HIV-1 packaging signal and rev response element(s). Antisense Res. and Dev. 4:19-29, Joshi, S., et al. 1991. Inhibition of human immunodeficiency virus type 1 multiplication by antisense and sense RNA expression. J. Virol. 65:5524-5530, Kim, J. H., et al., 1996. Inhibition of HIV replication by sense and antisense Rev Response Elements in HIV-based retroviral vectors. J. Acquir. Immune Defic. Syndr. 12:343-351, Meyer, J., et al., 1993. Inhibition of HIV-1 replication by high-copy-number vector expressing antisense RNA for reverse transcriptase. Gene 129:263-268, Renneisen, K., et al 1990. Inhibition of expression of human immunodeficiency virus-1 in vitro by antibody-targeted liposomes containing antisense RNA to the env region. J. Biol. Chem. 265:16337-16342 and Rhodes, A., et al. 1990. Inhibition of human immunodeficiency virus replication in cell culture by endogenously synthesized antisense RNA. J. Gen. Virol. 71:1965-1974), RNA decoys (Lee, T., et al. 1994. Inhibition of human immunodeficiency virus type 1 in human T cells by a potent Rev-response element decoy consisting of the 13-nucleotide minimal Rev-binding domain. J. Virol. 68:8254-8264 and Sullenger, B. A., et al 1990. Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication. Cell 63:601-608), and ribozymes (Ojwang, J. O., et al 1992. Inhibition of human immunodeficiency virus type 1 expression by a hairpin ribozyme. Proc. Natl. Acad. Sci. USA. 89:10802-10806 and Zhou C., I. Bahner, et al 1994. Inhibition of HIV-1 in human T lymphocytes by retrovirally transduced anti-tat and rev hammerhead ribozymes. Gene. 149:33-39).
The trans-dominant HIV-1 protein RevM10 was first evaluated in a clinical trial using genetically modified peripheral blood lymphocytes (Woffendin, C et al. 1996. Expression of a protective gene prolongs survival of T cells in human immunodeficiency virus infected patients. Proc. Natl. Acad. Sci. USA. 93:2889-2894), although recently a ribozyme (Leavitt, M. C., et al 1996. Ex vivo transduction and expansion of CD4+ lymphocytes from HIV+ donors: prelude to a ribozyme gene therapy trial. Gene Ther. 3:599-606) and a transdominant Rev and antisense TAR based (Morgan R. A et al 1996. Clinical protocol: Gene therapy for AIDS using retroviral mediated gene transfer to deliver HIV-1 antisense TAR and transdominant Rev protein genes to syngeneic lymphocytes in HIV-1 infected identical twins. Hum. Gene Ther. 7:1281-1306.) approach have received RAC and FDA approval.
Intracellular expression of antisense RNAs offers an attractive, alternative gene therapy approach to inhibit HIV-1 replication. Antisense RNAs have been described as very specific and efficient inhibitors in both prokaryotic and eukaryotic systems . Viral replication has been successfully inhibited by addition of in vitro synthesized antisense oligonucleotides or intracellularly expressed antisense RNAs . Inhibition of HIV-1 replication has been shown previously using antisense RNAs targeted against several viral regulatory (Chatterjee et al 1992, Joshi et al 1991, Kim et al 1996, Sczakiel, G. et al 1991. Inhibition of human immunodeficiency virus type 1 replication in human T cells stably expressing antisense RNA. J. Virol. 65:468472 and Sczakiel, G et al 1992. Tat- and Rev-directed antisense RNA expression inhibits and abolishes replication of human immunodeficiency virus type 1: a temporal analyses. J. Virol. 66:5576-5581) and structural gene products (Choli et al 1994, Gyotoky, et al 1991, Meyer et al 1993 and Rhodes et al 1990). A few reports described long antisense sequences expressed either intracellularly using retroviral vectors (Choli et al 1994, Gyotoky, et al 1991 and Rhodes et al 1990) or using antibody-targeted liposomal delivery (Renneisen et al). The different inhibition levels observed in these reports may reflect variation in antisense RNA expression levels, or secondary and tertiary RNA structures, which can influence the hybridization kinetics between two complementary RNAs (Sczakiel, G., M. Homann, and K. Rittner. 1993 Computer-aided search for effective antisense RNA target sequences of the human immunodeficiency virus type 1. Antisense Res. and Dev. 3:45-52), influencing the biological activity.
Generally, these efforts have targeted the early phase transcription (e.g., tat or rev genes) or have targeted RNA processing or initiation of translation in the late phase. Shorter antisense sequences have been favored due to the perceived risk of the antisense sequence folding to form a secondary structure with itself. To date, these efforts have not met with significant success.
It is now surprisingly discovered that the best target for antisense therapy is the full length or single-spliced RNA transcript. Antisense sequences which bind to multiple-spliced transcripts for a gene are less effective, probably because binding to the smaller transcripts results in fewer antisense molecules being available for the binding to the full length or single spliced transcripts. Moreover, longer sequences directed to the full length transcript (e.g., sequences greater than 600 base pairs, preferably greater than 1000 base pairs) are surprisingly effective and, contrary to the suggestion in the art, do not appear to form undesirable secondary structures.
Hereinafter we present the results of the antiviral activity of sequences complementary to the pol, vif, env genes and 3""LTR in HIV-1 infection experiments using a human CD4+T cell line (CEM-SS) and peripheral CD4+T lymphocytes (PBLs). Retroviral vectors are constructed expressing chimeric RNAs containing 1,100-1,400 nt long complementary HIV-1 sequences. The most efficient inhibition of HIV-1 replication is observed with an antisense sequence complementary to the HIV-1 env gene both in the CEM-SS cell line and in PBLs. This strong antiviral effect is further demonstrated in high inoculation dose infection experiments where reduction of the HIV-1 mRNAs correlates with low level of Gag and Tat protein production indicating that antisense RNA acts early during HIV-1 replication. Comparing the anti-HIV-1 efficacy of the antisense RNAs to the well documented (Bevec, D.,et al. 1992. Inhibition of human immunodeficiency virus type 1 replication in human T cells by retroviral-mediated gene transfer of a dominant-negative rev trans-activator. Proc. Natl. Acad. Sci. USA 89:9870-9874, Escaich, S., et al 1995. RevM10-mediated inhibition of HIV-1 replication in chronically infected T cells. Hum. Gene Ther. 6:625-634, Malim, M. H., et al. 1992. Stable expression of transdominant rev protein in human T cells inhibits human immunodeficiency virus replication. J. Exp. Med. 176:1197-1201 and Nabel, G. J., et al. 1995. A molecular genetic intervention for AIDSxe2x80x94effects of a transdominant negative form of Rev. Hum. Gene Ther. 5:79-92) trans-dominant RevM10 protein demonstrates the potency of the antisense mediated inhibition of HIV-1 replication.
It has further been discovered that antisense sequences to the gag, env, and pol, especially the env and pol portions of the full length transcript are particularly effective.
The above mentioned antisense constructs are particularly useful for providing gene therapy to patients suffering from HIV-1 infection, e.g., by transducing the HIV-1-susceptable cells of such patients, e.g., CD4+ cells or cells which are progenitors of CD4+ cells, e.g., hematopoietic stem cells (for example CD34+/Thy-1+cells), with the antisense constructs of the invention, so that the transduced cells and their progeny are resistant to HIV-1 infection.
The antisense constructs of the invention are suitably prepared by incorporating a wild-type HIV-1 gene or gene fragment into a vector in reverse orientation with respect to its promotor so that when the gene is incorporated into the genome of the host cell and transcribed, the opposite strand of the DNA is transcribed, producing a messenger RNA transcript which is complementary to the mRNA from the wild-type gene or gene fragment and will anneal with it to form an inactive RNA-RNA duplex, which is subject to degradation by cellular RNases.
Transduction of the HIV-1 susceptable cells using the antisense vectors can be carried out in vivo or ex vivo, but is suitably carried out ex vivo, by removing blood from the patient, selecting the target cells, inoculating them with a vector containing the antisense construct of the invention, and reintroducing the transduced cells into the body. By natural selection, the transduced HIV-1 resistant cells will replace the native HIV-1 susceptible cells, thereby enabling the patient to overcome the infection and regain immunocompetence. Alternatively, the patient receives non-autologous CD4+ cells or progenitors of CD4+ cells from a compatable donor which cells have been transduced with the antisense construct of the invention.
The invention thus provides:
1. A nucleic acid sequence which, when stably integrated into a human cell, is capable of generating mRNA which anneals e.g., under in vivo conditions, with a mRNA transcript from an HIV-1 provirus encoding env, env and pol or env, pol and gag and which is at least 0.6 kb, preferably at least 1 kb in length, most preferably 1-2 kb, e.g. from 1.1 to 1.5 kb; and which is selected from:
(i) a sequence which is antisense to the 1.4 kb fragment from the Apa1 cleavage site at ca. base 2004 of an HIV-1 provirus to the Pflm 1 cleavage site ca. base 3400 of an HIV-1 provirus, e.g. which is antisense to the sequence in FIG. 1 (SEQ. ID. NO. 1);
(ii) a sequence which is antisense to the 1.2 kb fragment from the Pflim 1 cleavage site ca. base 3400 of an HIV-1 provirus to the EcoR1 cleavage site ca. base 4646 of an HIV-1 provirus, e.g. which is antisense to the sequence in FIG. 2 (SEQ. ID. NO. 2);
(iii) a sequence which is antisense to the 1.3 kb fragment from the ApaL1 cleavage site ca. base 6615 of an HIV-1 provirus to the Bsm1 cleavage site ca. base 8053 of an HIV-1 provirus, e.g., which is antisense to the sequence in FIG. 3 (SEQ. ID. NO.3); and
(iv) a sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 99%, homologous to a sequence according to (i), (ii), or (iii) and which is capable of generating mRNA which annealss to the same mRNA transcript as that hybridizing to mRNA generated by (i), (ii), or (iii).
It is understood that the nucleic acid described in 1 above will be in RNA for when in a retroviral vector and will be converted to DNA upon incorporation of the provirus into the target cell. It is intended that both the RNA and DNA forms of the constructs are included within the scope of the invention.
The invention further provides
2. A vector comprising an antisense sequence according to 1 above.
The vector may be any vector capable of transducing a human hematopoietic cell, for example, an ecotropic, xenotropic, amphotropic or pseudotyped retroviral vector, an adeno-associated virus (AAV) vector, or an adenovirus (AV) vector. Preferably, the vector is a retroviral vector, preferably a vector characterized in that it has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), or murine embryonic stem cell virus (MESV), or for example, a vector from the pLN series described in Miller and Rosman (1989) BioTechniques 7, pp. 980-986. The antisense sequence replaces the retroviral gag, pol and/or env sequences. The promotor controlling expression of the antisense may be a strong viral promoter, for example MoMLV LTR.
The range of host cells that may be infected by a retrovirus or retroviral vector is generally determined by the viral env protein. The recombinant virus generated from a packaging cell can be used to infect virtually any cell type recognized by the env protein provided by the packaging cell. Infection results in the integration of the viral genome into the transduced cell and the consequent stable expression of the foreign gene product. The efficiency of infection is also related to the level of expression of the receptor on the target cell. In general, murine ecotropic env of MoMLV allows infection of rodent cells, whereas amphotropic env allows infection of rodent, avian and some primate cells, including human cells. Xenotropic vector systems utilize murine xenotropic env, and also allow infection of human cells. The host range of retroviral vectors may be altered by substituting the env protein of the base virus with that of a second virus. The resulting, xe2x80x9cpseudotypedxe2x80x9d virus has the host range of the virus donating the envelope protein and expressed by the packaging cell line. For example, the G-glycoprotein from vesicular stomatitis virus (VSV-G) may be substituted for the MMLV env protein, thereby broadening the host range. Preferably the vector and packaging cell line of the present invention are adapted to be suitable for transduction of human cells. Preferably, the vector is an amphotropic retroviral vector, for example, a vector as described in the examples below.
Optionally, the vector may contain more than one antisense sequence according to 1 above, e.g., two different antisense sequences, for example to pol and env, as described in the examples below.
Preferably, the construct lacks the retroviral gag, pol and/or env sequences, so that the gag, pol and env functions must be provided in trans by a packaging cell line. Thus, when the vector construct is introduced into the packaging cell, the gag-pol and env proteins produced by the cell assemble with the vector RNA to produce replication-defective or transducing virions that are secreted into the culture medium. The virus thus produced can infect and integrate into the DNA of the target cell, but generally will not produce infectious viral particles since it is lacking essential viral sequences. The packaging cell line is preferably transfected with separate plasmids encoding gag-pol and env, so that multiple recombination events are necessary before a replication-competent retrovirus (RCR) can be produced. Suitable retroviral vector packaging cell lines include those based on the murine NIH/3T3 cell line and include PA317 (Miller and Buttimore (1986) Mol. Cell Biol. 6:2895; Miller and Rosman (1989) Bio Techniques 7:980), CRIP (Danos and Mulligan (1988) Proc. Natl Acad Sci USA 85:6460), and gp+am12 (Markowitz et al. (1988) Virology 167:400); and also cell lines based on human 293 cells or monkey COS cells, for example ProPak A packaging cells, e.g., as described in Pear et al. (1993) Proc. Natl. Acad. Sci. USA 90:8392-8396; Rigg et al., (1996) Virology 218; Finer, et al. (1994) Blood 83:43-50; Landau, et al. (1992) J. Virol. 66:5110-5113. Retroviral vector DNA can be introduced into packaging cells either by stable or transient transfection to produce retroviral vector particles.
The antisense constructs of the invention have the further advantage that they will not interfere with expression of HIV inhibitory proteins, e.g., transdominant mutant proteins corresponding to the early phase short MRNA transcripts, for example mutants of tat or rev. Expression of such transdominant mutant proteins is useful in treating HIV infection because the mutant proteins interfere with the function of the wild-type HIV proteins and so inhibit HIV replication. A transdominant mutant protein of particular interest is RevM10, the use of which is described e.g., in Escaich, et al. Hum. Gene Ther. (1995) 6:625-634, and in WO 90/14427. Previously, co-expression of HIV antisense and transdominant mutant proteins was considered impractical because it was expected that the antisense would interfere with expression of the mutant protein. Using the antisense constructs of the invention, co-expression of the antisense with the transdominant mutant protein is not only feasible but provides a synergistic inhibition of the HIV by interfering with the virus at different stages of its replication cycle.
Thus the invention provides in a further embodiment:
3. A retroviral vector according to 2 above (i.e., comprising an antisense sequence according to 1 above) and further comprising a gene for an HIV-1 inhibitory protein, e.g., a gene for a transdominant mutant form of tat or rev, especially the gene for RevM10.
Packaging cell lines comprising the vectors according to 2 or 3 above, e.g, as described above, are also within the scope of the invention.
The invention also provides in a further embodiment:
4. A cellular composition comprising at least one human hematopoietic cell (e.g. CD4+ cell or progenitor of CD4+ cells, e.g., a stem cell, e.g., a CD34+/Thy-1+ cell) stably transduced with an antisense sequence according to 1 above and optionally additionally transduced with a gene for a transdominant mutant form of tat or rev, especially RevM10, e.g., transduced with a vector according to 2 or 3, supra, e.g., for use in a method according to 5 below;
The invention also provides in a further embodiment:
5. A method for treatment of HIV-1 infection in a subject in need thereof comprising
isolating hematopoietic cells (e.g. CD4+ cells or progenitors of CD4+ cells, e.g., stem cells, e.g., CD34+/Thy-l+ cells) from said patient;
transducing said cells with an antisense sequence according to 1 above, and optionally additionally or simultaneously transducing said cells with a gene for an HIV-1 inhibiting transdominant mutant form of tat or rev, especially RevM10, e.g., transducing said cells with a vector according to 2 or 3, supra; and
reintroducing the transduced cells into the patient.
The invention also provides in a further embodiment:
6. The use of an antisense sequence according to 1 above or a vector according to 2 or 3 above in the manufacture of a cellular composition according to 4 above or in a method of treatment according to 5 above.